The amyloid precursor protein (APP) is a type 1 transmembrane protein that has been implicated in the onset of Alzheimer's Disease (AD). The biological role of APP is not well understood, leading to many questions concerning its role in AD pathogenesis. It is known that APP is subject to alternative pathways of proteolytic processing; one pathway involves the enzymes and ?-secretase and produces benign fragments. The other pathway involves the enzymes and ?-secretase and the resulting fragment is believed to cause Alzheimer's Disease. This product is commonly referred to as the A peptide. We study the transmembrane protein that is the precursor of the A peptide, a 99 amino acid transmembrane protein C99. The hallmark of AD is the formation of A aggregates, or plaques. These plaques ultimately precipitate, resulting in disrupted neuronal function. In addition, the precursors to the A plaque can be cytotoxic to neuronal cells. The proposed experiments will focus on a structure and function approach to studying AD. Determination of the three dimensional structure of C99 is a crucial step in understanding how APP functions and interacts with other proteins. The three dimensional structure of C99 will be determined using Nuclear Magnetic Resonance (NMR) Spectroscopy in lyso-myristoyl- phosphoglycerol. In previous studies in our lab, we have shown that C99 can specifically bind the cholesterol analogue -cholbimalt[5]. We believe that this interaction is physiologically relevant as patients who have AD have elevated levels of cholesterol in their brain. Interestingly, epidemiological studies have shown that people who regularly take statins have a decreased prevalence of AD[13]. To better understand this interaction and its implications for disease, I will determine th three dimensional structure of C99 bound to cholesterol in a DHPC/DMPC bicelle lipid mimetic. The structure of the C99/cholesterol complex will reveal information regarding cholesterol's role in regulation of and secretase cleavage. To further understand how cholesterol regulates APP and C99 processing, we will use fluorescence confocal microscopy to study the membrane localization of APP and C99 under varying levels of cholesterol. The fluorescence studies will show how the interaction between cholesterol and APP alters APP membrane localization, demonstrating new information regarding the regulation of the secretase cleavage pathway. Taken in conjunction with our structural studies, this novel information will open a new avenue for potential AD therapeutics via regulation of the interaction between APP, C99, and cholesterol. PUBLIC HEALTH RELEVANCE: The proposed studies will contribute to a better understanding of the interaction between the Amyloid Precursor Protein (APP) cleavage product C99 and cholesterol and how this interaction possibly regulates Alzheimer's Disease (AD). The cleavage of C99, which occurs in cholesterol rich domains, results in the release of the A peptide, which is highly prone to aggregation and is believed to cause AD. These studies will allow for a better understanding of how the interaction between C99 and cholesterol structurally and functionally regulates AD.